Purification of antibiotics



Patented Dec. 29, 1953 UNITED STATES PATENT OFFICE PURIFICATION OF ANTIBIOTICS No Drawing. Original application August 9, 1947, Serial No. 767,852. Divided and this application December 8, 1950, Serial No. 199,922

8 Claims. (Cl. 260-210) This application is a division of our application Serial No. 767,852, filed August 9, 1947, now Patacid; and (4) decomposing the precipitate. [The term primary streptomycin-containing liquid nt No. 2,537,934, dated January 9, 1951. omprehends, inter alia; (a) the culture liquid This invention relates to basic antibiotics of obtained by growing Streptomyces grzseus under the streptomycin type, i. e., to members of the 5 conditions and in a mediumsuitable for the progenus composed of streptomycin and antiduction of streptomycin, and removing the solids biotically-active basic compounds which (like from the medium; (12) the culture liquid of en streptomycin) are capable of forming water-solhanced potency obtained by acidification of such uble salts with acids such as sulfuric and waterculture (with hydrochloric or sulfuric acid, for insoluble salts with organic base-precipitating example), the liquid being neutralized; and (a) reagents (e. g., derivatives of streptomycin, such the liquid obtained by acid-extraction of the as 'dihydrostreptomycin, and similarly-acting solids separated from such culture, the liquid antibiotics, such as streptothricin). being neutralized] I 1944, Schatz, Bugie, and Waksman (Proc. It is the object of this invention to provide soc. Exp. Biol. Med. 1944, 57, 244) showed that a 15 s mpl fli i t and th rw ad a a us potent antibiotic, termed streptomycin,- was methods of purifying basic antibiotics of the formed during the growth of .the organism Acstreptomycin typeespecial1y streptomycin; and tinomg ces griseus (now 7 called Streptomyccs it is a further object of this invention to provide griseus); and this antibiotic has since been erta n salt-type derivatives of basic antibiotics demonstrated to be of high clinical utility. Of the streptomycin type useful in these purifica- It was later found that a number of streption methods and for other purposes, and methtomycins are formed at the same time. The first ods of preparing these derivatives. streptomycin obtained in pure crystalline form It has been found that basic antibiotics of the (as a reineckate) and fully characterized (Win streptomycin typ interact With u face-active tersteiner and Fried application, Serial No. a ents of the or anicallyi u ed polybasic- 666,541, filed May 1, 1946, now Patent No. inorganic-acid type to form certain salt-type 2,501,014, dated March 21, 1950) is now referred combinations which are much less soluble in t as streptomycin A; and. the second strep water than the antibiotics and may be recovered; tomycin characterized (Fried and Titus applicaand it has also been found that such salt-type tion Serial No. 737,400, filed March 26, 1947, now an erivatives of the antibiotics may be decomposed Patent'No. 2,565,653, dated August 28, 1951) is to eCOVeI the antibiotics. now referred to as streptomycin B. Moreover, The methods of this invention essentially comthere are indications that still other streptomyprise i t racting a basic antibiotic of the strep-- cins are formed, at the same time and/or may be tomycln type (especially t ep o ycin) with a formed at the same time by changes in the culurface-active agent of the organically substituted ture conditions; and it is intended that each of poly-basic-inorganic-acid type in a solvent for such antibiotics and any i t thereof the reactants (especially Water). The formed (whether in the form of the free base or Water pe Comb nation of the antibiotic and the soluble n; thereof) be comprehended by t surface-active agent is relatively insoluble in term streptomycin when employed unmodified 40 Water. d ay be recovered by filtration. centrifugation or other suitable means when water is employed as the solvent for the reactants. When the employed solvent for the reactants is one (such as methanol) in which the salt-type combination is soluble, the formed salt-type combination is recovered by removing the solvent (and purifying the residue, as by washing with hereinafter.

Streptomycin has been purified heretofore by various methods, all of which were complex and/or inefiicient (and consequently expensive). For example, a method widely employed prior to this invention essentially comprised the following steps: 1) treating a primary streptomycin-con taining liquid with an activated charcoal, which 3?: 2 1 5 gfigg g g ig gfi g g l gf fig selectlvely adsqrbs the Streptomycm (2) elutmg 5 0 purification methods of this invention essentially the stmptomycm from the chargoa'l Wlth an aque' comprise treating an impure basic antibiotic of ous, water-soluble mineral acid, preferably at a t Streptomycin type with a surface active agent l htly elevated temperature (say about 3050 of the organically-substituted polybasic-in- C.) (3) treating the eluate with an organic-b'ase- 1- ani -a i .t in l t for t t t precipitating reagent, preferably phosphotungstic 06 especially treating an aqueous solution of the impure antibiotic (e. g., a primary sterptomycincontaining liquid, or an aqueous solution of a partially-purified streptomycin, such as the eluate referred to hereinbefore) with the surfaceactive agentrecovering the formed salt-type combination of the antibiotic and the surface.

active agent, and converting the latter combination into a Water-soluble salt ofth'e; antfiiiotic.

Such conversion may be effected, for example, by dissolving the salt-type derivative irra solvent therefor (such an methanol), treating. the solution with an aqueous, water-soluble; relativelystrong acid (especially with an aqueous, water:-

soluble, relatively-strong mineral acid), and recovering the formed water-soluble salt (of the basic antibiotic of the streptomycin type), for example, by adding a miscible nonsolvent'forthe water-soluble salt (such as acetone), and recov ering the precipitate. The product thus obtained is considerably purer. than the antibiotic treated, and the recovery of antibiotic activity in the purification treatment is of a high. order. By the practice of this invention it is possible to obtain uniformly high yields of relatively-pure streptomycin (for example) having. a potency above about 400 units/ mg.

Among the preferred surface-active agents of the organically-substituted. polybasic-inorganicacid type for the purposes. of. this invention are those of the formula R-OXO-Y wherein R is the residue of a substantially water-immiscible organic hydroxyl compound, -OX--O- is the divalent acid-residue of av Water-soluble polybasic-inorganic-acicl' (e. g., sulfuric or phosphoric acid), and Y is a member oi the group consisting' of H and cations forming'water-soluble salts with the anion RJ-OX-O-.. Notable among such wetting agents for the purposesv of this invention are those in which the pol'ybasic-inorganic-acid is sulfuric, and the acid is partially esterified with a higher aliphatic alcohol,. i. e., wetting agents of the group consisting of mono (higher-aliphatic) esters of sulfuric acid, and water-soluble salts thereof.

Other preferred surface-active agents of the organically-substituted polybasic-inorganic-acid typefor the purposes of this invention are those of'the group consisting of aromatic sulfonic acids, sul'fonated oils, sulfonated higher fatty" acid derivatives, and water-soluble salts thereof.

Among the partial higher-alkyl esters of sulfuric acid utilizable in the practice of this invention are: the group of sodium salts of sulfates of synthetic higher aliphatic alcohols, such as C4159 CH $21-15) C2H4CH SOLN a) C2H4CH (C2115) 2v [e-. g., Tergitol Penetrant 7'] C4H9CH ('CzI-Is) CzH iCl-I SO4Na) CHzCH (CH3 7 2 Le. g., Tergitol Penetrant a], and.

CiH'sCH (CzHe) CH2SO4N'a Le. g., Tergitol Penetrant 08]; and the series of partial. sulfuric-acid esters of higher aliphatic alcohols and their salts, such as sodium octyl sulfate, sodium oleyl sulfate, sodium cetyl sulfate, sodiumv stearyl sulfate, and sodium lauryl sulfate [e. g., Aurinol, Wetanol, the Duponols, and the Gardinolsl. Among the aromatic sulfonic acids,sulfonated oils, and sulfonated higher fatty acid derivatives. utilizable in the practice of this invention are: the sodium s-ulfonates ofv higher fatty acid. esters and amides, such as the sodium. salt of sulfonated ethyl (or other alkyl) oleate [e-. g., Igepon AP Extra], and

[e. g., Igepon T]; the sodium sulfonates of petroleum hydrocarbons [e. g., Ultrawetl; the sodium salt of a polyalkyl benzene sulfonic acid having ten carbon atoms [e. g., Ultrawet, 40 A], and other sodium alkyl aryl sulfonates [e. g., Nacconol NRSF]; and Turkey red oil (1. e., sulfonated castor oil). Among the partial esters of phosphoric acids utilizable in the practice of this invention are dicresyl phosphate, lecithin, and a phosphorated higher alcohol of the formula ('capryhsNasPsOzo ['e. g., W. A. 58].

Among the water-soluble, relatively-strong acids utilizable for recovery of the antibiotics from their salt-type combinations with the surface-active agents are sulfuric, hydrochloric, phosphoric, oxalic, citric, sulfamic, and nitric.

For maximum efficiency, the amount of the surface-active agent employed should be substantially that required to combine with all the antibiotic in the solution treated, the optimum amount of surface-active agent being therefore dependent on the concentration of the antibiotic solution treated and the potency of the antibiotic. By using the stoiehiometric amount of surfaceactive agent (with respect to the antibiotic activity of the solution treated) one can obtain a.

tomycin type is treated with an aqueous solutionoi the surface-active agent, and. the precipitate formed is recovered and converted into a water soluble salt of the antibiotic. Alternatively, the surface-active agent may be added to the antibiotic solution in solid form; or both the antibiotic and the surface-active agent may be added in solid form. to water (or other solvent for the reactants).

The salt-typecombination of the antibiotic and the. surface-active agent may separate from the solvent for the reactants in various forms; e. g.,. as an oily, waxy or gelatinous material, or may not separate at all but remain dispersed in the solvent (alltheseinsoluble forms being comprehended by the. term precipitate as employed.

herein); and depending on its form, the. precipitate is. separated. by filtration, centrifugation, treatment. with a filter-aid followed by filtration,

or other conventional means known to thoseskilled in the art.

The precipitation of the salt-type combination of the antibiotic and the surface-active agent is affected by the concentration of they reactants in, and the pH. of, the reaction medium-maximum.

precipitation being obtained when the concentra-- tion is high, and the pH is between about 5 and about; 8, and preferably around. 7.

Alternatively, thesalt-type combination of thethe antibiotic-rather than purification of the antibiotic-is the objective, one may employ a reconstituted aqueous solution of the antibiotic (e. g., an aqueous solution of the highly purified or pure antibiotic).

The conversion of the salt-type combination of the antibiotic and the surface-active agent, into a water-soluble salt of the antibiotic may be effected in the following ways, inter alia: by dissolving the salt-type combination in a solvent therefor (e, g., methanol), treat ng the solution with a water-soluble, relatively-strong acid, and recovering the formed water-soluble salt of the antibiotic (e. g., by adding a miscible non-solvent for the water-soluble salt, such as acetone) by contacting the salt-type combination (in solution) with an adsorbent (e. g., alumina) or anionexchange resin which has been pretreated with the desired water-soluble, relatively-strong acid, and recovering the formed water-soluble salt of the antibiotic from the treated solution (e. g., by removing the solvent); by dissolving the salttype combination in a substantially water-immiscible organic solvent for soaps (e. g., n-butanol or refined fermentat on-amyl-alcohol), intimately contacting the solution with an aqueous, water-soluble, relatively-strong acid, recovering the aqueous phase, and drying it (preferably freeze-drying; i. e., freezing and subjecting to a high vacuum to sublime oiT the water) by dissolvingv the salt-type combination in a solvent therefor in which the desired water-soluble salt is insoluble (e. g., methanol-acetone mixture), treating the solution with an aqueous, watersoluble, relatively-strong acid, and recovering the precipitated water-soluble salt of the antibiotic; by treating a solution of the salt-type combination with an aqueous, water-soluble, relatively strong acid in the presence of an anion-exchange resin; or by various other means of effecting in timate contact and interaction of the salt-type combination with the water-soluble, relativelystrong acid.

The following examples are illustrative of the vention (all solutions or dilutions referred to without identification of the solvent or diluent being solutions in or dilutions with water) Example 1 (a) To 500 ml. of a streptomycin-containing [e. g., Tergitol Penetrant '7] is added with agitation, and the agitation is continued for a half hour. The oil which separates is a salt-type combination of streptomycin and the ,surfaceactive agent, and contains about 94% of the culture-filtrate activity.

(b) A salt-type combination thus obtained from a culture filtrate batch of 1,245,000 units activity is dissolved in methanol, reprecipitated once by adding an equal volume of water, redissolved in 80% methanol, and passed through a column of alumina which has previously been washed with hydrochloric acid to pH 4.1, and dried. The percolate (about 250 m1., including methanol wash) is collected; and the streptomycin hydrochloride is recovered by adding an equal volume of water, distilling off the methanol under vacuum, removing any undecomposed salttype combination, adjusting the pH of the remaining aqueous solution to 5.5, and freeze-drying. About 1.55 g. streptomycin hydrochloride having a potency of 320 units/mg. is thus obtained (a yield of about 40%).

Example 2 0.5 g. of a partial sulfuric-acid ester of a higher aliphatic alcohol [e. g., Duponol C] in 20 ml. water is added to 500 ml. of a steptomycincontaining eluate having apotency of 300 units/ml. and a pH of 6.5 [obtained by treating a streptomycin-containing culture filtrate with an activated charcoal, and eluting the streptomycin from the charcoal with dilute hydrochloric, nitric or sulfuric acid], the cloudy solution is agitated for one hour, and the flocculent precipitate formed is filtered off. The thusobtained salt-type combination of streptomycin and the surface-active agent contains about 76% of the eluate activity; it may be used as a thera-- peutic agent per se, or converted into a watersoluble salt of streptomycin; e. g., as described in Example 6 hereinafter.

Example 3 (a) 16 ml. of a 25% solution of C4H9CH C2H5) C2H4CH (SO4Na) C2H4CH(C2H5) 2 [e. g., Tergitol Penetrant 7] is added with stirring over a period of one hour to 7 liters of a streptomycin-nitrate-containing eluate having a potency of 195 units/ml. and a pH of 7.0; and the precipitate formed is allowed to age for 12 hours at about 5 0., and filtered off. The thusobtained salt-type combination of streptomycin and the surface-active agent contains about 93% of the eluate activity.

(b) A salt-type combination thus obtained from an eluate batch of 850,000 units activity is pressed or otherwise treated to remove entrained water, and dissolved in suflicient -100% methanol (depending on the water-content of the thus partially-dried salt-type combination) to make up a solution in ml. of 90% methanol; and the solution is passed through a 2.5 x 25 cm. column of anion-exchange resin [e. g., Amberlite IR-4B; of. U. S. Patent 2,402,384, dated June 18, 1946], which has previously been conditioned with dilute hydrochloric acid to pH 2.5. The percolate is collected in two ml. portions (including methanol wash); and each portion is treated by adding an equal volume of water, distilling ofi the methanol under vacuum, adjusting the pH of the remaining aqueous solution to 5.5 by treatment with a neutral anion-exchange resin, filtering, and freeze-drying. The first and second portions of the percolate yield, respectively, about 0.8 g. streptomycin hydrochloride having a potency of about 679 units/mg, and about 0.1 g. having a potency of about 425 units/mg. (total yield about 73.1%).

(2): alternative) A salt-type combination thus obtained from an eluate batch of 860,000 units activity (weighing about 4 g.) is dissolved in ml. 90% methanol; and the solution is agitated with 50 g. of an anion-exchange resin which pre- [e. g., Tergitol Penetrant ,7l1is added to a solution of 1 g. streptomycin hydrochloride having apoitencygo'f' 525-unitsj/mg; in 100 ml. water, themixture is shakenahalf -ho.ur on .a shaking machine,

and-the waxy precipitate formed. is filtered oil. The salt-type combination of streptomycin and the 7' suria-ceeactive; agent thus obtained (Weighing -aboutlizg.) containsabout-{96.5970 of theactivity ;of th streptomycin hydrochloride solution treated.

" Earcmple 15 solution of .1- g. .ofw- 3,:-..-pa37.tl3.-1-; sulfuric-acid gester. ofta higher-aliphatic alcohol. [e. g.,-Duponol C]-,in 50-.ml.=water. isradded Ito a-solution of g. streptomycin hydrochloride, having .a' potency of 580.11nits/rng. -in.20 ml. Water, and the thick ge- I rlatinous mixture. formed. is..,added to A00 ml, cold water. The gelatino us precipitate formed, is-collected by centrifugation, washed by re-suspending in Water and collecting by centrifugation, and.

dried in vacuo.

The salt-type combination of streptomycin and the surface-active agent thus obtainedhas;a-potency,eta-about 35,0 units/me, is insoluble in Water, soluble in substantially water- "immiscible .organic :solvents. for soaps (e. g., re-

:ifined .fermentation-amylealcohol) and gives :ggoodfidispersions in-oily .media' (e-g peanut oil, qQlt: ethyl .oleate) havingclinical utility.

"Example :6

; 1.5;; g: ofa salt-,ty

ei combinatiomofi streptomy- :cin and: a partialsulfuricracid estenof .a higher aliphatic alcohol, obtained,aforqexample, :as de- 1 scribed n Example 2 (the-streptomycin compo- .nent. of which has an activity of 376,000 units,;1as determined-by, chemical; assay) .is dissolved in,

-concentratedhydrochloric acid is; then added followed by an additional resultingprecipitate (streptomycin hydrochloride) .is filtered off and dissolved in water .:,and the,- solution; -is-; adjusted; to

:.pl-I 63.8.133 addition ;of neutral anioneexchange v.:resin, .-and. freezeedried. About 0.'l;-g.- streptomycin hydrochloride having, a;;poten ;y-:of: about 425 .units/mgais thus obtained. (Yield; about 79%,.)

-Erample 7 p .1280; m1. of aL 25,.%.-. solutionroira partial sulfuricacidesteriof a higher.- aliphaic; alcohol [e.- g., Dup.on ol;'Cl isslowly. added toga solution of 50 g.

par'tiallymurifiedstr distilled .water, units/ml; and the eptomycin. sulfate in 24liters" having a potency of 1000 .white fiocculent precipitate formed isisolatedrbyVcentrifugation, and dried .-in vacuo (10 mm.)

hours. The salt-typ at room temperature for 12 e combination thus obtained inza yield of about 57,; .,.has a potency-of about -224 units/mg andis suitable for clinical use --,either. per, se :or in. various pharmaceutical forms.

Using.310 ml. ,of

a. clarified (carbon treated) ,..surface-active. agent ..in1..the;foree0in p dur 3 a gsalt-type combination having 7 a potency of about 293 units/male obtained in a yield of. about Example .8

A solution of g. of a partial sulfuric-acid ester of a higheraliphatic alcohol [e. g., Duponol C] in 350 ml. water, having a pI-I of'Qfl, is added -with stirring to a solution of 30 g. streptomycin sulfate havinga potency of 490 units/mg. and a pH- of 5.2 and the solidwhich' precipitates is separated by centrifugation, and washed with water by centrifugation. The salt-type combination thus-"obtained is resuspended in 400 ml.' water, and the suspension is freeze-dried, yielding about 38.5%. of a-vvhite fiuiiy solid-having a-potencyoi -320-units/mg. (On washingivith -Water, resuspending, in1water; antifreeze-drying the suspension, the lastwtraces of inorganic saltaare' -removed.)

The -salttype combination 1 thus obtained is only slightly soluble in-waterpan'd disperses well in peanut oil, ethyl oleate, and other oily media.

A smooth dispersion suitable for intramuscular injection and having -a potency of $200,000 units/ml. ma be obtained, for example-by adding slowly, with stirring, 1 ml. peanut oil (oret-hyl oleate) to 666mg. 0f:the-sa1ttype combination.

le. g., Tergitcl Pentrant '7]- is .addedwith agitation to la-solution of 05- g. dihydrostreptomycin sulfate in 250 ml. .vvater, havingapotency of 1210 units/ml; and: the precipitated :salt-type: combination (containingover 83% of the activity of the; solutiontreated) is recovered by filtration.

Similar treatment offan aqueoussolution of ,streptothricin hydrochloride having apotency of 500 units/mg. yields the corresponding salttype derivative ofrstreptothricin.

Example 10 .40.,ml. concentrated hydrochloric acid is added andthe mixture, is stirred vigorously;-and-ml. .ivateris added and stirring continued forlOminutes. ,The aqueous layerpformed on separation is recovered, ,neutralizedivith aneutral anion- --exchange resin [e. g.,-Amberlite lit-.4113], and-filtered. The filtrate (andwash), having a total volume of about 190 ml., has a potency or about 65,000 units/ml. (and about 85% recovery. [Washing of the'methylamylacetate layer with ml. .viater, neutralization ofthe wash, and filtration yieldsadditional filtrate hayinga potency of 16,000 units/ml. (the total recovery being about 97%). The solid streptomycin hydrochloride obtained on freeze-drying has .a potency of about 575 units/mg.

Example 11 .A streptomycin -,,containing culture filtrate having apotency, of. 21 1,units/m1. and a pH of 1.5 is treated Withl g. .ofMan activated carbon [e.;g., Darco S-5,1l,per' 20,000 units, and. filtered; and thefiltrate is adjusted to pH 6.5-7.0. A 25% solution of Example 12 A streptomycin hydrochloride having a histamine content of 0.1 mg. for each 207 units streptomycin activity is converted into a salt-type combination with a surface-active agent as described in section (a) of Example 3; and the salt-type combination is decomposed as described in section (b) of that example. The thus-obtained streptomycin hydrochloride has a histamine content of 0.1 mg. for each 610 units streptomycin activity.

' Example 13 g. of a salt-type combination of streptomycin and surface-active agent obtained as described in section (a) of Example 1 (the streptomycin component ofv which has an activity of 4,240,000 units) is dissolved in 100 ml. methanol; and a slurry of g. of an anion-exchange resin [e. g., Amberlite IR-4B] in 300 ml. water, having a pH of 6.5, is added. Then, while agitating and maintaining the mixture at -55" 0., concentrated hydrochloric acid is added dropwise at such rate that the pH is maintained at 3.0-3.5. (The endpoint of the decompositon is determinale' by following the increase of maltol precursor in the aqueous solution.) After an hour (during which about 3.5 ml. concentrated hydrochloric acid has been added), the pH is allowed to rise to'5.0-5.5; the solution is filtered; the methanol is removed from the filtrate by evaporation in vacuo; and the residual solution is freeze-dried, yielding about 6.5 g. streptomycin hydrochloride having a potency of about 578 units/mg. (about 90% yield).

i Example 14 E 15' g. of a salt-type combination of streptomycin and surface-active agent obtained as described in section (a) of Example 1 (the streptomycin component of which ha an activity of 2,960,000 units) is idssolved in 100 ml. methanol; and dry HCl is passed into the solution until no more precipitation occurs. The mixture is then diluted with 500 ml. acetone; and the precipitate is filtered oif and dissolved in 100 ml. water. The pH of the solution is adjusted to 5.5 by addition of a neutral anion-exchange resin [e. g., Amberlite IR-4B]; the solution is filtered; and the filtrate is freeze-dried, yielding about 4.96 g. streptomycin hydrochloride having a potency of about 385 units/mg. (a yield 01 about 64.5%).

Example 15 (a) 1000 gals. of a streptomycin-containing culture filtrate having a potency of 400 units/ml. and a pH of 3-4, is heated to 20 C., and neutralized to pH 7.5102 by adding 10% sodium hydroxide solution (about 4.5 gals. being required) and the neutralized solution is treated with 251 lbs. activated carbon [e. g., Darco G-GO] and 20 lbs. filter aid [e. g., Hyfiol. The slurry is stirred for an hour and filtered; and the carbon cake is washed with at least 100 gals. water, and blown dry with air.

251 gals. of the second eluate from a previous elution, such as described hereinafter, is made up to a volume of 753 gals. by addition of tap water; the carbon cake described in the preceding paragraph is added; the slurry is maintained at 35-40 0., while stirring; and the pH of the suspension is adjusted to 22:02 by addition of 10% nitric acid (about 14.1 lbs. being required). After stirring for an hour, the carbon is removed by filtration and blown dry, and the filtrate (first eluate) is collected.

The once-extracted carbon is added to 251 water for a second elution,

gallons fresh tap which is efiected as described in the preceding paragraph, a small amount of 10% nitric acid being added (if necessary) to re-adjust the pH to2.2- -0.2. The thus-obtained second eluate is used for the first elution described in the preceding paragraph. The total yield from the culture filtrate is about 82%.

The first eluate is adjusted to pH 7.5101 with 10% sodium hydroxide solution (about 4.2 gals. being required), while maintaining the temperature at 10 C. or below; 7.5 lbs., of a filter aid [e. g., Hyfio] is added; and the slurry is filtered. The colorless (iron-free) filtrate, at 10-15 C., is chargedv with half the total volume of a 25% solution of [e. g., Tergitol Penetrant 7] required for precipitation of the streptomycin (the amount being based on the activity of the eluate, a determined by chemical assay, in the ratio of 1 ml. per 92,400 units streptomycin). After this portion of the surface-active agent has been added, the testing for completeness of precipitation is begun (a sample of the slurry being filtered and about 2 ml. of filtrate being collected; and 1-2 drops 50% phosphotungstic acid being added to the filtrate, which forms a precipitate if the precipitation by the surface-active agent was incomplete). More of the solution of surface-active agent is then charged in one-liter portions, until the test shows complete precipitation (about 28.7 lbs., or 3.4 gals. of a 25% solution ofthe surface-active agent being required for complete precipitation). The batch is stirred for a half hour, and the solid (salt-type combination) recovered by centrifuging. The yield from the culture filtrate is about 73.8%, the precipitation elliciency being about b) The wet salt-type combination is dissolved in 90% methanol at 20-25 C., in a ratio of 15 g. per ml. solvent (over six gals. methanol being required); the solution is filtered; the filtrate is charged slowly with sufficient concentrated hydrochloric acid to decompose the salttype combination and lower the pH to 08:01 (about 0.9 lbs. acid being required); and the mixture is drowned in 194 lbs. acetone, and 'allowed to stand for an hour at 5-10 C. The precipitate (streptomycin hydrochloride) is filtered as dry as possible on a vacuum filter, and then dissolved in water, using a ratio of 100 ml. water per 10 g. of the salt-type combination treated (about six gals. water being required). The solution is neutralized by stirring with about 1.09 lbs. neutral anion-exchange resin [e. g,, Amber- 6.5, to adjust the pH of the streptomycin hydrochloride to its most stable pH range of 6.0-6.5. The resulting mixture is filtered; the filtrate is distilled free of methanol and acetone at 15-20 mm., and filtered; and the filtrate is freeze-dried. The yield of streptomycin hydrochloride thus obtained from the culture 11 filtrate is about 51.8%, the-decomposition'yield being about 70%.

[The neutral anion-exchange resin employed may be prepared as follows: change resin (e. g., Amberlite IR-4B) is added to gals. tap' water; sufficient 10%- sodium carbonate solution is added to adjust the pH to 10.5102; and the alkaline resin is washed with distilled water until the pH of the aqueous. medium reaches 6.5. The neutral resin is stored in the aqueous medium until employed for neutralization; i. e., is filtered from the-aqueous medium just before it is to be used] (1)": Alternative) combination is dissolved in absolute methanol at 2U-25 C., in the ratio of g. of the salt-type combination per 100' ml. 100% methanol (at least 9 gals. methanol being required) the solution is nltered; 45.5 lbs. of a neutral (pl-I 6.5) anion-exchange resin [e. g., Amberlite IR-4B] is-added to the filtrate; and sufficient wateris added to the resulting slurry to make a methanol medium (about27 gals.- water being. required). The slurry (volume about 42 gals.) is heated to -55 C-., and maintained at that temperature while 0.5 gal. concentrated hydrochloric acid is added at of the mixture is maintained between 3,0 .and.3.5. Then the mixture is stirred until the pI-I rises to its. maximum, between is .below6, small amounts of neutralanion-exchange resin are added until the; final pH is between'6 and 6.5). and the resinis washed with suflicient water to remove. any adhering streptomycin hydrochloride; the combined filtrate and wash (volume about,!l7.5 gals, having a potency of about 6,505 units/ml.) is then concentrated'to about 3 gals; and, the streptomycin hydrochloride is recovered by freeze-dry ng. The decomposition: yield, is 60-80%.

The surface-active. agents employed. in. the.

foregoing examples maybe replacedby equivalent amounts of other surface-activeagents of the organically-substituted polybasicginorganic-acid' type (exemplificative subgenera and species ofw Among other which. are namedhereinbeiore) salt-type .derivatives of streptomycin obtainable inaccordance, with this invention and especially suitable; for therapeutic use per sepharmaceutical forms) areithose obtainable with B, dihydrostreptomycin A, dihydrostreptomycin.

B, and streptothricin.

The basic antibiotic oi the streptomycintype purified by the method of this inventionmay be further purified by repetition purified or further purified by anyother method, especially byone of the following: (I') intimately contacting an aqueous solution ganic solvent for the carboxylic acid, recovering thelorganic solvent salt-type derivative of the antibiotic therein-into 10 lbs. of anion-ex-.

15.2 lbs. oithe dry salt-type:

such rate that'the pH.

5.6 and 6.2 (iii the pH,

The slurry is then filtered,

(and in.

of the same -puri-, fication method. Also, it may be preliminarily of the antibiotic with a substantially water-insoluble carboxylic acid and a substantially water-immiscible or phase, and converting the awater-soluble salt of the antibiotic; (chap-- plication Serial No. 762,205, filed July 19; 1947f now Patent No. 2,644,816, dated July 7, 1953); (II) treatingan aqueous solution of the anti biotic with a water-soluble salt of a substantially water-insoluble 'carboxylic acid, recovering: the precipitated salt-type combination of the antibioticand the carboxylic acid, and converting it into a water-soluble salt of the antibioticlcf. application Serial No. 762,206, filed July 19, 1947, now Patent No. 2,631,143, dated March 10, 1953) and (III) intimately contacting an aqueous solution of the antibiotic with a surface-active agent of the organically-substituted polybasic-inor ganic-acid-type and a substantially water-immiscible organic solvent for soaps, recovering the'organic solvent phase, and converting-the salt-type derivativeof the antibiotic therein into a water-soluble salt of the-antibiotic (cf. application Serial No. 767,851, filed August 9, 1947; now Patent No. 2,537,933, dated January 9, 1951).

The invention may be variously otherwise embodied within the scope of the appended claims.

Weclaim:

1., The method which comprises interactingan antibiotic of the group consisting. of streptomycin, dihydrostreptomycin, and streptothricin with a water-soluble member. of the group con sisting-oi sulfonates of higher fatty acid esters, sulfonates or" higher i2 tty acid amides, sulfonates. of petroleum hydrocarbons, polyalkyl-Iaryl sulfonates, and sulfonated castor oil. in a solventffor: the reactants.

2. The method which comprises, interacting streptomycin with-a water-soluble member. of the,

water-soluble member of the group consisting. l of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum. hydrocarbons, polyalkyl-aryl sulfonates,- and sulfonated castor oil;in1a;solvent for thereactants, and recovering theformed salt-- type, combination of the antibiotic and. the.

sulfonate.

4. The meth'odoi purifying an antibioticof the group consisting of streptomycin, dihydrostreptoa mycin;and.streptothricinwhich comprises treatingan impure-member of said 'groupuwithca. water-soluble member or" sulfonates oihigher fatty acid esters,-. sulfonatesofhigher fatty acidamides, sulfonates-of. pe-.

troleum hydrocarbons, polyalkyl aryl sulfonates,

and sulfonated castor oil in-a solvent for the reactants, recovering the 4 formed salt-type com bination of the antibiotic and-the sulfonate, and converting the latter combination into a watersoluble salt of the antibiotic.

5 The-method of purifying streptomycin; whichv comprises treating an impure streptomycin with: group consisting:

a' water-soluble member'ofthe of sulfonates of higher fatty acid: esters, .sulfonztes-ofhigher fatty acid amides,sulfonatespi" petroleum hydrocarbons, polyalkylarylsufonates, and sulfonated castor oil; in water, recovering the insoluble salt-type-combination orstreptomycin and the sulfona-te; and converting the latter combination into a water-soluble-salt of streptomycin.

higherfatty acid amides,

the group consisting of.

6. The method which comprises treating an antibiotic of the group consisting of streptomycin, dihydrostreptomycin, and streptothricin with a water-soluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil, in a solvent for the reactants, the sulfonate being in stoichiometric amount with respect to the antibiotic activity of the solution, and recovering the insoluble salttype combination of the antibiotic and the sulfonate.

7. The method which comprises treating a water solution of an antibiotic of the group consisting of streptomycin, dihydrostreptomycin, and streptothricin with a water solution of a watersoluble member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher fatty acid amides, sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil, and recovering the insoluble salt-type combination of the antibiotic and the sulfonate.

fatty acid amides,

member of the group consisting of sulfonates of higher fatty acid esters, sulfonates of higher sulfonates of petroleum hydrocarbons, polyalkyl-aryl sulfonates, and sulfonated castor oil, and recovering the insoluble salt-type combination of the antibiotic and the sulfonate.

A.- LOTT. JACK BERNSTEIN. LEON J. HEUSER.

References Cited in the file of this patent UNITED STATES PATENTS Number Name a Date 2,462,175 Folkers Feb. 22, 1949 2,501,014 Wintersteiner et a1. Mar. 21, 1950 2,537,933 Lott et a1. Jan. 9, 1951 2,537,934 Lott et a1. Jan. 9, 1951 2,538,847 Regna et a1 Jan. 23, 1951 

1. THE METHOD WHICH COMPRISES INTERACTING AN ANTIBIOTIC OF THE GROUP CONSISTING OF STREPTOMYCIN, DIHYDROSTREPTOMYCIN, AND STREPTOTHRICIN WITH A WATER-SOLUBLE MEMBER OF THE GROUP CONSISTING OF SULFONATES OF HIGHER FATTY ACID ESTERS, SULFONATES OF HIGHER FATTY ACID AMIDES, SULFONATES OF PETROLEUM HYDROCARBONS, POLYALKYL-ARYL SULFONATES, SULFONATED CASTOR OIL IN A SOLVENT FOR THE REACTANTS. 